Of the human serum proteome in HUPO Plasma Proteome Project 222

Xiaohai Li, Yan Gong, Ying Wang, Songfeng Wu, Yun Cai, Ping He, Zhuang Lu, Wantao Ying, Yangjun Zhang, LiyanJiao, Hongzhi He, Zisen Zhang, Fuchu He, Xiaohang Zhao and Xiaohong Qian

10.1 Introduction 222

10.2 Materials and methods 223

10.2.1 Materials 223

10.2.2 Human serum samples 223

10.2.3 Integrated strategy for characterising analytical approaches 223

10.2.4 Depletion ofthe highly abundant serum proteins by MARS 224

10.2.5 Desalting and concentrating the flow-through fractions by centrifugal ultrafiltration 224

10.2.6 Fractionation of depleted serum samples by anion-exchange HPLC 225

10.2.7 Protein fractionation by 2-D HPLC with nonporous RP-HPLC 225

10.2.8 The 2-DE strategy for the analysis of serum proteins 226

10.2.8.2 In-gel digestion via automated workstation 227

10.2.8.3 Protein spot identification by MALDI-TOF-MS/MS 227

10.2.9 Shotgun strategy for the analysis of serum proteins 228

10.2.9.1 Trypsin digestion of serum proteins 228

10.2.9.2 Protein identification by micro2-D LC-ESI-MS/MS 228

10.2.9.3 Data processing 229

10.2.10 Protein fractionation strategy for the analysis of serum proteins 229

10.2.10.1 2-D LC fractionation of serum proteins 229

10.2.10.2 Digestion of the 2-D LC separated fractions 229

10.2.10.3 1-D microRP-HPLC-ESI-MS/MS identification of digested serum proteins 230

10.2.11 Offline shotgun strategy for the analysis of serum proteins 230

10.2.11.1 Offline SCX for first-dimension chromatographic separation of peptides 230

10.2.11.2 1-D capillary RP-HPLC/microESI-IT-MS/MS analysis for the SCX-separated peptide fractions 231

10.2.12 Optimised nanoRP-HPLC-nanoESI IT-MS/MS for the reanalysis of offline SCX-separated peptides (offline-nanospray strategy) 231

10.3 Integrated analysis of the whole data sets 231

10.3.1 Protein grouping analysis 231

10.3.2 Sequence clustering 232

10.4 Results and discussion 233

10.4.1 Depletion ofthe highly abundant serum proteins 233

10.4.2 The 2-DE strategy for the analysis of serum proteins 233

10.4.3 2-D HPLC fractionation for the analysis of serum proteins 234

10.4.4 Shotgun strategy for the analysis of serum proteins with online SCX 237

10.4.5 Shotgun strategy for the analysis of serum proteins with offline SCX 237

10.4.6 Offline SCX shotgun-nanospray strategy for the analysis of serum proteins 239

10.4.7 Comparison of the five strategies for the analysis of the human serum proteome 241

10.5 Concluding remarks 246

10.6 References 246

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